CN116920177A - 一种小檗碱/软骨细胞/可注射水凝胶仿生材料及其制备方法和用途 - Google Patents
一种小檗碱/软骨细胞/可注射水凝胶仿生材料及其制备方法和用途 Download PDFInfo
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- CN116920177A CN116920177A CN202310842443.4A CN202310842443A CN116920177A CN 116920177 A CN116920177 A CN 116920177A CN 202310842443 A CN202310842443 A CN 202310842443A CN 116920177 A CN116920177 A CN 116920177A
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- Prior art keywords
- berberine
- chondrocyte
- injectable hydrogel
- cartilage
- hydrogel
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Abstract
本发明属于生物医学技术领域,涉及一种小檗碱/软骨细胞/可注射水凝胶仿生材料及其制备方法和用途。本发明突破性的将磷酸修饰甲基丙烯酸酐化壳聚糖可注射水凝胶(支架材料)、软骨细胞(种子细胞)、小檗碱(生物活性物质)三者结合为一形成小檗碱/软骨细胞/可注射水凝胶仿生软骨支架材料。该材料能够实现小檗碱对软骨细胞的长时间作用,制备成软骨支架,可作用于其周围的残存软骨细胞及新生软骨细胞,在水凝胶提供的攀附物质基础上实现软骨组织的再生及修复,并且起到“1+1+1>3”的作用,对软骨缺损治疗提供新策略。
Description
技术领域
本发明属于生物医学技术领域,涉及一种小檗碱/软骨细胞/可注射水凝胶仿生材料及其制备方法和用途。
背景技术
关节软骨是一种由软骨细胞和细胞外基质构成的覆盖于关节表面的透明软骨,主要起到润滑和减震作用,由于缺少血管和神经,容易因创伤、肿瘤、炎症等造成损伤后难以愈合。随着医疗及科技的进步,我国及世界人口老龄化日趋严重,软骨缺损的患者也越来越多,这不仅影响了患者生活质量,造成残疾,给患者家庭甚至国家医疗带来了沉重负担,也因此关节软骨损伤修复对骨科医生而言是一个棘手问题,受到越来越多的关注。只有使关节软骨形态学、生物学及理化性质均恢复成接近正常软骨,才能够重建关节功能,这既是临床医务工作者的机遇,对其而言也是挑战。
医用材料是近些年新兴的一种医学和材料学的交叉学科引领下迅速发展起来的一种材料,支架材料、种子细胞、生物活性物质是医用材料的三要素,三合为一的性能良好的医用材料受到越来越多的关注。建立功能优异的仿生软骨材料,并进行成果转化,成为医疗领域促进软骨缺损修复的一条新途径,如果研发得当,将会造福广大患者。
医用材料的基础是支架材料,良好的医用支架材料要有良好的生物安全性、生物相容性、生物可降解性,并且材料获取方便、成本相对较低等特点。以壳聚糖为基材的水凝胶在软骨缺损中起到重要的作用。壳聚糖是由甲壳素脱去乙酰基后产生的物质,而甲壳素分布非常广泛,在自然界中含量居第二位,资源丰富,容易获得。壳聚糖不溶于水和普通的有机溶剂,其改性后被广泛用于医学领域。壳聚糖本身是由2-10个氨基葡萄糖构成的寡糖,在生物体内容易降解,具有免疫调节、抗氧化、抗自由基等特点,降解后的成分对生物体无毒害。此外,壳聚糖在吸水后可以膨胀,有较强的吸水性,用壳聚糖制成的生物支架有利于细胞的粘附、增殖和分化,细胞可以更好的在其表面生长,形成具有生物功能的复合体。
种子细胞也与软骨缺损关系密切,根据其是否分化可以分为分化后种子细胞(如软骨细胞、成骨细胞、成纤维细胞等)和未分化种子细胞(如BMSCs、ADSCs、滑膜间充质干细胞(SMSCs)等)。在软骨损伤修复中,最重要的细胞是软骨细胞,其本身就是软骨组织中的唯一细胞,而且其分泌的II型胶原和aggrecan也是软骨损伤修复的必要物质。
生物活性物质在软骨缺损修复中起到重要作用,其种类多种多样,比如天然药物类物质小檗碱。近些年的研究显示,小檗碱在软骨缺损修复中起到重要作用,其可以通过调控相关信号通路中的靶因子表达而起到促进软骨缺损修复的作用。小檗碱,也叫作黄连素,是从中药黄连中分离的一种季铵生物碱,也是黄连抗菌的主要成分。小檗碱本身味苦,呈黄色针状结晶,最初是由M.-E.夏瓦利埃和G.佩尔坦于1826年从Xanthoxylonclava树皮中获得,小檗碱的化学式为C20H18NO4,分子量为336.36,熔点204-205℃,密度为1.117g/cm3,在热水中可溶解,在水或乙醇中微溶,在三氯甲烷中极微溶,不溶于乙醚。小檗碱在自然界中分布广泛,大约有4个科10个属内有小檗碱存在。由于小檗碱属于植物抗菌素,其在骨科、胃肠道、心血管疾病中起到重要作用。
软骨缺损是骨科医生在临床上需要处理的棘手问题,可伴有局部疼痛及功能障碍,严重者导致残疾,对其研究迫在眉睫。现有的软骨缺损修复金标准是取自体或异体软骨移植,这样就存在二次手术、费用高、操作复杂、排异反应、患者痛苦大等不利因素。新型的支架材料、种子细胞和生物活性物质良好结合的仿生软骨材料在近年来备受关注,然而这种组合型仿生软骨材料在现有技术中应用极少,大多数都存在负载物质不能长时间作用、支架材料硬度不够或无法提供新生组织血管生长所需的孔隙空间、种子细胞无法诱导新生组织生成等劣势,安全可靠性也有待提升。
发明内容
本发明针对现有技术问题,提供了一种小檗碱/软骨细胞/可注射水凝胶仿生材料及其制备方法和用途。本发明突破性的将磷酸修饰甲基丙烯酸酐化壳聚糖可注射水凝胶(支架材料)、软骨细胞(种子细胞)、小檗碱(生物活性物质)三者结合为一形成小檗碱/软骨细胞/可注射水凝胶仿生软骨支架材料。该材料能够实现小檗碱对软骨细胞的长时间作用,制备成软骨支架,可作用于其周围的残存软骨细胞及新生软骨细胞,在水凝胶提供的攀附物质基础上实现软骨组织的再生及修复,并且起到“1+1+1>3”的作用,对软骨缺损治疗提供新策略。
本发明的技术方案如下:
首先,本发明提供一种小檗碱/软骨细胞/可注射水凝胶仿生材料,其技术方案为:将小檗碱(BBR)预处理过的ATDC5软骨细胞接种到可注射水凝胶形成的固体凝胶中;具体的制备方法,包括:
(1)小檗碱(BBR)预处理ATDC5软骨细胞:采用含有6-36μmol/L小檗碱以及10%FBS和1%双抗的DMEM/F12培养基刺激ATDC5软骨细胞12-36h,然后进行离心,获得预处理细胞;
(2)将ATDC5软骨细胞接种到可注射水凝胶形成的固体凝胶中:将可注射水凝胶注入注射器中形成固体凝胶块,切割成小块水凝胶后进行冻干,消毒后,放入孔板或培养皿中,将预处理细胞通过细胞计数板进行计数并制备细胞悬液(以10%FBS和1%双抗的DMEM/F12培养基来制备),以3-5×106细胞/mL浓度的100μL细胞悬液滴加在冻干后的固体凝胶块上。
其中,步骤(1)中刺激时间优选为16-32h,更优选24h。
步骤(1)所述的培养基中小檗碱的浓度优选为12-36μmol/L,更优选12-24μmol/L,最优选24μmol/L。
步骤(2)中细胞浓度优选5×106细胞/mL。
步骤(2)中切割成小块水凝胶优选为圆柱形,更优选切割为直径0.6cm、高0.4cm的圆柱形。
所述的可注射水凝胶可采用现有技术完成,尤其可以选择发明专利ZL202010574779.3所述的可注射抗压裂可降解超分子水凝胶,其对天然多糖进行甲基丙烯酸酐及磷酸或膦酸改性,复合含金属粒子形成超分子结合,制备可注射抗压裂水凝胶。在水凝胶中多功能团相互作用,如氢键、亲疏水作用、静电作用及范德华作用等及超分子结合的特点使该超分子水凝胶具有抗压裂性能,压缩到一定程度(压缩应变大于85%)而不发生碎裂,且在水溶液中浸泡一段时间后可恢复至接近初始的高度,其中磷酸根与含金属粒子可形成金属配体超分子结合,使该水凝胶具备可注射性。尤其与本发明中所述小檗碱(BBR)预处理过的ATDC5软骨细胞结合后,ATDC5软骨细胞在水凝胶上的活性良好,水凝胶携载ATDC5软骨细胞体外安全性显著。
本发明进一步保护上述小檗碱/软骨细胞/可注射水凝胶仿生材料在软骨支架方面的用途;
本发明所获得的仿生材料能够作为软骨支架为缺损处软骨组织提供小檗碱刺激后的微环境作用,以及小檗碱与软骨细胞结合的功能更加优异的软骨细胞,并通过水凝胶作为支架载体,填补软骨缺损的同时提供软骨细胞的攀附材料,并随着新生软骨组织的形成而支架材料逐步降解,最终形成完整的新生软骨组织,从而减少了取自体软骨修复缺损的二次手术感染风险、操作繁琐、供体软骨不足、异体或异种软骨存在的排异反应等,降低了软骨缺损患者治疗的痛苦、风险及费用。
附图说明
图1为对比实验例不同处理组膝关节软骨缺损手术后4W、8W、12W的总体形态图;
图2为对比实验例中不同处理组Micro CT的影像学评估及三维重建。
图3为对比试验例中术后12W不同处理组小鼠心、肝、脾、肺和肾的HE染色图。
图4为对比实验例中术后4W、8W、12W的HE染色的组织学评估。
图5为对比实验例中COL II在软骨损伤中的表达及相关量化分析。
图6为对比实验例中SOX9在软骨损伤中的表达及相关量化分析。
图7为对比实验例中AGC在软骨损伤中的表达及相关量化分析。
其中:
图1中,A组a,f,k;B组b,g,I;C组c,h,m;D组d,I,n;E组e,j,o。
图2中,*代表与A组相比;#代表与B组相比。*,P<0.05;**,P<0.01;***,P<0.001;****,P<0.0001;#,P<0.05;##,P<0.01;###,P<0.001;####,P<0.001。
图5中,*代表与A组相比;#代表与B组相比。*,P<0.05;**,P<0.01;***,P<0.001;##,P<0.01;###,P<0.001;####,P<0.001。
图6中,*代表与A组相比;#代表与B组相比。*,P<0.05;****,P<0.0001;#,P<0.05;##,P<0.01;###,P<0.001。
图7中,*代表与A组相比;#代表与B组相比。**,P<0.01;***,P<0.001;#,P<0.05;##,P<0.01;###,P<0.001;####,P<0.001。
具体实施方式
下面通过具体实施方式例对本发明进行详细描述。本发明的范围并不受限于该具体实施方式。
其中:
ATDC5软骨细胞购买于上海安为生物科技有限公司,使用含有10%胎牛血清和双抗的DMEM/F12培养基培养于含5%CO2,37℃的培养箱中。
以下实施例及对比例中涉及的实验技术未明确说明的,均采用下述方法完成:
细胞复苏
(1)准备好离心管、移液枪、枪头、培养皿等,预热水浴锅温度至37℃后,将培养基放入水浴锅中预热。
(2)从-80℃冰箱中取出冻存的ATDC5软骨细胞,将冻存管放入37℃水浴锅使冻存管中的内容物快速融化后,在超净工作台中打开冻存管,加入1mL培养基,用移液枪轻轻吹打混匀后转移至15mL离心管,再次加入3mL培养基,再次混匀后,放入离心机,以1500rpm离心3min。
(3)离心完成后,弃去上清液,向离心管中添加1mL培养基,将细胞沉淀轻轻吹打混匀后成细胞悬液,用移液枪将细胞悬液移入培养皿中,最后加入足量的培养基,轻轻摇晃细胞培养皿,使细胞均匀分布,把细胞放入37℃,5%CO2的培养箱中培养。
细胞冻存
(1)在显微镜下观察ATDC5软骨细胞生长密度达80%-90%时,用移液枪吸取培养皿里的培养基,加入预热好的PBS缓冲液,摇晃并清洗干净后,用移液枪吸去PBS,重复两次。
(2)往培养皿中加入预热好的胰酶2mL,充分摇匀后放入培养箱中孵育1-2min,显微镜下观察细胞,看到已被消化的细胞充分悬浮,向培养基内加入4mL预热好的培养基,轻轻吹打混匀后,用移液枪吸取细胞悬液移至15mL离心管中。1500rpm离心3分钟。
(3)离心完成后,尽量将上清液移除,加入1mL细胞冻存液,并用移液枪轻轻吹打,使细胞均匀分散在冻存液中,并将冻存液移至冻存管中,然后将冻存管放入-80℃冰箱中冷冻,待后续实验时使用。
CCK8实验:
(1)培养实验组和对照组细胞,用预热的胰蛋白酶消化细胞,预热的培养基终止消化,1500rpm离心3min,加入1mL培养基将细胞充分重悬,吸取10μL细胞悬液加入细胞计数板,用细胞计数仪计算细胞数量后,在96孔板内铺板,每组6个孔,每孔大约3000~5000个细胞。
(2)细胞贴壁后,按照LPS刺激分为4组(对照组、2μg/mL、4μg/mL、6μg/mL)、BBR刺激分为6组(对照组、3μmol/L、6μmol/L、12μmol/L、24μmol/L、36μmol/L)和LPS对ATDC5软骨细胞的最适作用浓度刺激下再次分组(对照组、LPS组、3μmol/LBBR+LPS、6μmol/L BBR+LPS、12μmol/L BBR+LPS、24μmol/L BBR+LPS、36μmol/LBBR+LPS)进行相应的处理,分别在LPS刺激后12、24、36、48h,BBR刺激后的24h,最适作用浓度刺激后的24h弃去培养基,向每孔中加入100μL的培养基和10μL CCK-8试剂10μL,将96孔板放入培养箱内继续孵育1h,在酶标仪上测定450nm处的OD值。
Calcein-AM/PI活死细胞染色实验
(1)培养实验组细胞和对照组细胞,用预热的胰蛋白酶消化细胞,培养基中和并终止胰酶消化,1500rpm离心3min,以每孔3000个细胞密度将细胞将细胞接种于96孔板中。显微镜下观察细胞后,根据CCK8筛选的LPS和BBR浓度作用于ATDC5软骨细胞。
(2)将Calcein-AM/PI试剂盒(同仁化学)从-20℃冰箱取出后在室温下进行复温。待充分融化后,避光条件下准备15mL离心管并加入5mL PBS缓冲液,再加入10μL Calcein-AM储存液和15μLPI储存液,混匀制成工作液。此时Calcein-AM的浓度为2μmol/L,而PI的浓度为4.5μmol/L。
(3)在BBR和LPS刺激作用后,避光下操作,弃去细胞培养基,用PBS反复清洗3次,加入Calcein-AM/PI工作液,在培养箱中孵育15min。
(4)孵育完后,使用在荧光显微镜下用不同激光检测细胞,活细胞为黄绿色荧光,死细胞为红色荧光。
可注射水凝胶制备(参照发明专利ZL202010574779.3):
(1)用烧杯装300mL去离子水,加入3mL乙酸溶液。称取3g壳聚糖并溶于上述乙酸溶液中。在烧杯中用搅拌子在磁力搅拌机上搅拌,加速壳聚糖溶解。待壳聚糖充分溶解后加入1.1mL MA,在室温下反应24h。
(2)称取20.787g MES溶解于150mL去离子水中,在称取1.17gPS溶解于上述MES溶液中。称取3.51g EDC和1.17g NHS溶解在上述PS溶液中。
(3)将(1)中得到的溶液逐滴滴加到(2)溶液中,反应在冰上进行。并在室温下反应24h。
(4)反应后,将所得溶液倒入透析袋中,并用夹子夹住透析袋。在去离子水中透析1周,每天换去离子水3次。
(5)将透析袋中的溶液倒入大皿中,并放入-80℃冰箱中冷冻,待完全冷冻后,将大皿放入冻干机中冻干。大约3天后得到冻干的CSMAP。
(6)按20g/mL称取CSMAP溶液与去离子水中,充分搅拌溶解后。按10mg/mL MA、0.4mg/mL BIS和1mg/mL I2959加入上述溶液中,充分搅拌完成后,用注射器抽取上述溶液,将注射器放入紫外光下交联成胶。
(7)交联完成后,将注射器放入-20℃冰箱冷冻,待冷冻后用小刀切成合适大小的水凝胶,放入去离子水中浸泡2天去除未能完全反应的化合物。最后放入-80℃冰箱冷冻,待冷冻后放入真空冻干机中,冻干后最终得到水凝胶。放入真空干燥箱中保存并用于后续实验。
实施例1
小檗碱/软骨细胞/可注射水凝胶仿生材料制备:
(1)小檗碱(BBR)预处理ATDC5软骨细胞:采用含有24μmol/L小檗碱的培养基刺激ATDC5软骨细胞24h,然后进行离心,获得预处理细胞;
(2)将ATDC5软骨细胞接种到可注射水凝胶形成的固体凝胶中:将可注射水凝胶注入2.5mL注射器中形成固体凝胶块,切割成直径0.6cm、高0.4cm的水凝胶后进行冻干,消毒后,放入孔板中,将预处理细胞以5×106细胞/mL浓度的100μL细胞悬液滴加在冻干后的固体凝胶块上。
实施例2-4
与实施例1制备方法相同,其中不同参数如下表所示:
对比例1
直接将ATDC5软骨细胞接种到可注射水凝胶形成的固体凝胶中:将可注射水凝胶形成的固体凝胶消毒后,放入24孔板中,将ATDC5软骨细胞以5×106细胞/mL浓度的100μL细胞悬液滴加在冻干后的固体凝胶块上。
对比实验例
从珠海百试通生物科技有限公司购买75只6周龄健康C57Bl/6小鼠,饲养在笼子里,接受水和食物喂养。动物造模(参考文献Bethan L Thomas,Suzanne E Eldridge,BabakNosrati,Mario Alvarez,Anne-Sophie Thorup,Giovanna Nalesso,Sara Caxaria,AidaBarawi,James GNicholson,Mauro Perretti,Carles Gaston-Massuet,CostantinoPitzalis,Alison Maloney,Adrian Moore,Ray Jupp,Francesco Dell'Accio.WNT3A-loaded exosomes enable cartilage repair[J].JExtracell Vesicles,2021,10(7):e12088.),使用异氟烷对小鼠进行麻醉,将小鼠固定于手术台上,对右膝关节备皮,使用碘伏消毒右膝关节3次,于右侧关节处显露髌韧带,从内侧将髌韧带切开,将髌骨脱位并充分暴露膝关节,使用手钻(直径大约0.8mm)在右膝关节股骨髁非负重区钻孔,钻孔深达软骨至软骨下骨出血,缺损直径大约为0.8mm,深1mm。将小鼠随机分为A组72只、A’组3只,并进一步将A组72只小鼠随机分到B、C、D、E组各18只,并进行如下修复处理后,进行常规饲养,并在4、8、12W收集B、C、D、E组各6只大鼠的膝关节标本进行相关观察实验,A’组仅在12W时用于体内安全性分析的正常对照。
A组为正常对照组,72只小鼠的左侧膝关节仅切开滑膜囊后不做任何处理行假手术后缝合。
A’组不做任何处理,常规饲养。
B组小鼠的右侧膝关节股骨髁非负重区手钻钻孔,钻孔深达软骨至软骨下骨出血,缺损直径大约为0.8mm、深1mm(为软骨缺损组)。
C组小鼠在B组基础上仅植入可注射水凝胶填补软骨缺损部位。
D组小鼠在B组基础上植入对比例1制备的材料填补软骨缺损部位。
E组小鼠在B组基础上植入实施例1制备的仿生材料填补软骨缺损部位。
(1)大体标本观察
术后4周,B组(图1b)缺损处有少量组织填充,但仍存在缺损。C组D组可观察到一些组织,但仍可见缺损未完全修复,C组缺损处不规则,而D组较C组(图1c和d)再生组织多,E组(图1e)缺损周围有透明样的软组织增生,但边缘仍可见缺损区域。
术后8周,B组(图1g)有轻微修复,但仍存在凹面。C组(图1h)缺损周围可见半透明样组织增生,可见缺损边界,D组(图1i)修复组织填充缺损处,但表面不规则。E组(图1j)缺损处修复组织与周围组织明显连接,边界模糊。
术后12周,B组(图1l)有组织修复现象,但局部仍可见缺损。C组(图1m)已基本填充缺损区域,但边界仍清晰可见。D组(图1n)表面接近光滑,与周围组织接近相同。E组(图1o)修复组织与周围类似。
(2)Micro CT评估
相关骨参数的变化随着治疗的干预发生了改变,Micro CT的代表性图像显示于图2中。如图2所示,在4W时,E组BV/TV、Tb.N、Tb.Th均较其他组高且更接近A组,而Tb.Sp在B、C、D、E组中呈逐渐降低的趋势。在8W和12W时也表现出了相同的结果趋势。因此说明,我们的新型材料可能诱导了软骨下骨的重建,进而在软骨缺损修复中起到重要作用。
(3)体内安全性分析
为验证水凝胶植入小鼠膝关节后对小鼠体内脏器是否有影响。我们切取了12W各组小鼠的心、肝、脾、肺和肾并进行HE染色,以A’组作为正常对照组。如图3所示,结果表明,小鼠的心、肝、脾、肺和肾未见明显的异常。说明水凝胶在小鼠体内具有良好的生物安全性。
(4)HE染色对体内软骨再生的评估
HE染色用于观察小鼠股骨软骨组织的结构。如图4:
4W时E组中软骨缺损中随着时间的延长缺损接近修复,有组织填充,软骨下骨重建,但仍存在间隙。在B组中,缺损未显示愈合无组织填充,C组和D组见纤维组织修复。
8W时E组有软骨下骨重建,组织区域修复。B组有少量纤维组织填充,C组有纤维组织填充但仍存在一定缺损,D组修复组织填充较为致密,与边缘相对接近。
12W时E组中有组织生长,缺损有修复趋势,软骨下骨重建,表面接近光滑。B组缺损仍然存在。C组细胞分布紊乱,组织松散,有软骨下骨重建。D组软骨下骨重建,结构未见明显缺损。
(5)免疫组织化学染色
采用免疫组织化学染色检测软骨相关蛋白的表达情况,具体检测方法为:(1)将染色所需的切片放入烤箱中烘烤6小时,并将切片放至室温冷却后用于染色;(2)切片常规二甲苯、梯度酒精脱蜡至水;(3)用免疫组化笔在距离待测的组织区域3mm处画圈,避免画到组织上;(4)在免疫组化笔圈定的区域滴加胰蛋白酶抗原修复液,在37℃温箱中孵育30-40min;(5)切片放入PBS洗3次,每次5min。在圈定的区域滴加内源性过氧化物酶阻断剂,孵育10min;(6)切片放入PBS洗3次,每次5min。在圈定的区域滴加非特异性染色阻断剂,孵育10min;(7)切片放入PBS洗3次,每次5min。在圈定的区域添加相应的一抗(SOX9、AGC、COLII),4℃冷库孵育过夜;(8)切片放入PBS洗3次,每次5min。在圈定的区域添加生物素标记的羊抗小鼠/兔、小鼠、兔IgG聚合物或兔抗羊IgG聚合物,孵育10min;(9)切片放入PBS洗3次,每次5min。在圈定的区域添加链霉菌抗生素蛋白-过氧化物酶,孵育10min;(10)切片放入PBS洗3次,每次5min。除去PBS,加入DAB显色。显色后,放入PBS中终止显色;(11)苏木素复染2min,盐酸分化2-3s。自来水冲洗返蓝。常规逐级乙醇梯度脱水,二甲苯透明。中性树脂封片。
其中COLII、SOX9、AGC蛋白的表达量越高,说明软骨组织修复越好并且越接近正常软骨。
如图5所示,在4W、8W、12W检测COL II的表达:4W时D和E组COL II的表达较B组高,差异具有统计学差异。8W时E组的表达较B组高,差异具有统计学差异。12W时,C、D和E组与B组相比表达增加,且均具有统计学差异。
如图6所示,在4W、8W、12W检测SOX9的表达:在4W时C和E组SOX9的表达较B组高,差异具有统计学差异。8W时E组的表达较B组高,差异具有统计学差异。12W时,D和E组与B组相比表达增加,具有统计学差异。
如图7所示,在4W、8W、12W检测AGC的表达:在4W时D和E组AGC的表达较B组高,差异具有统计学差异。8W时C、D和E组的表达较B组高,差异具有统计学差异。12W时,D和E组与B组相比表达增加,具有统计学差异。
综上所述,本发明小檗碱/软骨细胞/可注射水凝胶仿生材料能够作为软骨支架为缺损处软骨组织提供长期的小檗碱刺激作用后的微环境,以及小檗碱与软骨细胞结合的功能更加优异的软骨细胞,并通过水凝胶作为支架载体,填补软骨缺损的同时提供软骨细胞的攀附材料,并随着新生软骨组织的形成而支架材料逐步降解,最终形成完整的新生软骨组织,从而降低了软骨缺损患者治疗的痛苦、风险及费用,避免了取自体或异体软骨的弊端。
尽管已描述了本发明的优选实施例,但本领域内的技术人员一旦得知了基本创造性概念,则可对这些实施例作出另外的变更和修改。所以,所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (10)
1.一种小檗碱/软骨细胞/可注射水凝胶仿生材料,其特征在于,具体的制备方法,包括:
(1)小檗碱(BBR)预处理ATDC5软骨细胞:采用含有6-36μmol/L小檗碱的10%FBS和1%双抗的DMEM/F12培养基刺激ATDC5软骨细胞12-36h,然后进行离心,获得预处理细胞;
(2)将ATDC5软骨细胞接种到可注射水凝胶形成的固体凝胶中:将可注射水凝胶注入注射器中形成固体凝胶块,切割成小块水凝胶后进行冻干,消毒后,放入孔板或培养皿中,将预处理细胞进行计数并制备细胞悬液,以3-5×106细胞/mL浓度的100μL细胞悬液滴加在冻干后的固体凝胶块上。
2.根据权利要求1所述的一种小檗碱/软骨细胞/可注射水凝胶仿生材料,其特征在于,步骤(1)中刺激时间为16-32h。
3.根据权利要求1或2所述的一种小檗碱/软骨细胞/可注射水凝胶仿生材料,其特征在于,步骤(1)中刺激时间为24h。
4.根据权利要求1所述的一种小檗碱/软骨细胞/可注射水凝胶仿生材料,其特征在于,步骤(1)所述的培养基中小檗碱的浓度为12-36μmol/L。
5.根据权利要求1或4所述的一种小檗碱/软骨细胞/可注射水凝胶仿生材料,其特征在于,步骤(1)所述的培养基中小檗碱的浓度为12-24μmol/L。
6.根据权利要求1或4所述的一种小檗碱/软骨细胞/可注射水凝胶仿生材料,其特征在于,步骤(1)所述的培养基中小檗碱的浓度为24μmol/L。
7.根据权利要求1所述的一种小檗碱/软骨细胞/可注射水凝胶仿生材料,其特征在于,步骤(2)中细胞浓度优选5×106细胞/mL。
8.根据权利要求1所述的一种小檗碱/软骨细胞/可注射水凝胶仿生材料,其特征在于,步骤(2)中切割成小块水凝胶为圆柱形。
9.根据权利要求1或8所述的一种小檗碱/软骨细胞/可注射水凝胶仿生材料,其特征在于,步骤(2)中切割成小块水凝胶为直径0.6cm、高0.4cm的圆柱形。
10.权利要求1-9任意一项所述的小檗碱/软骨细胞/可注射水凝胶仿生材料在软骨支架方面的用途。
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